Govender Kamini, Naicker Tricia, Lin Johnson, Baijnath Sooraj, Chuturgoon Anil Amichund, Abdul Naeem Sheik, Docrat Taskeen, Kruger Hendrik Gerhardus, Govender Thavendran
Catalysis and Peptide Research Unit (CPRU), School of Health Sciences, University of KwaZulu-Natal, E-block, 6th Floor, Room E1-06-016, Westville Campus, Durban, South Africa.
School of Life Sciences, University of KwaZulu-Natal, Durban, South Africa.
AMB Express. 2020 Mar 10;10(1):43. doi: 10.1186/s13568-020-00969-w.
Insulin has captured researchers' attention worldwide. There is a rapid global rise in the number of diabetic patients, which increases the demand for insulin. Current methods of insulin production are expensive and time-consuming. A PCR-based strategy was employed for the cloning and verification of human insulin. The human insulin protein was then overexpressed in E. coli on a laboratory scale. Thereafter, optimisation of human insulin expression was conducted. The yield of human insulin produced was approximately 520.92 (mg/L), located in the intracellular fraction. Human insulin was detected using the MALDI-TOF-MS and LC-MS methods. The crude biosynthesised protein sequence was verified using protein sequencing, which had a 100% similarity to the human insulin sequence. The biological activity of human insulin was tested in vitro using a MTT assay, which revealed that the crude biosynthesised human insulin displayed a similar degree of efficacy to the standard human insulin. This study eliminated the use of affinity tags since an untagged pET21b expression vector was employed. Tedious protein renaturation, inclusion body recovery steps, and the expensive enzymatic cleavage of the C-peptide of insulin were eliminated, thereby making this method of biosynthesising human insulin a novel and more efficient method.
胰岛素已引起全球研究人员的关注。全球糖尿病患者数量迅速上升，这增加了对胰岛素的需求。目前的胰岛素生产方法既昂贵又耗时。采用了一种基于聚合酶链式反应（PCR）的策略来克隆和验证人胰岛素。然后在实验室规模上，人胰岛素蛋白在大肠杆菌中过表达。此后，对人胰岛素表达进行了优化。所产生的人胰岛素产量约为520.92（毫克/升），存在于细胞内部分。使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和液相色谱-质谱（LC-MS）方法检测人胰岛素。使用蛋白质测序对粗生物合成蛋白序列进行验证，其与人胰岛素序列具有100%的相似性。使用MTT法在体外测试人胰岛素的生物活性，结果表明粗生物合成的人胰岛素显示出与标准人胰岛素相似的疗效程度。由于使用了无标签的pET21b表达载体，本研究无需使用亲和标签。省去了繁琐的蛋白质复性、包涵体回收步骤以及胰岛素C肽昂贵的酶切步骤，从而使这种生物合成人胰岛素的方法成为一种新颖且更高效的方法。
